Agrobacterium-mediated transformation methods have been used principally in dicotyledonous plants. Agrobacterium-mediated transformation in dicotyledons facilitates the delivery of larger pieces of heterologous nucleic acid as compared with other transformation methods such as particle bombardment, electroporation, polyethylene glycol-mediated transformation methods, and the like. In addition, Agrobacterium-mediated transformation appears to result in relatively few gene rearrangements and more typically results in the integration of low numbers of gene copies into the plant chromosome.
Monocotyledons are not a natural host of Agrobacterium. Although Agrobacterium-mediated transformation has been reported for asparagus (Bytebier B., et al. Proc. Natl. Acad. Sci. USA 84:5354-5349, 1987) and for Dioscore bublifera (Schafer et al. Nature 327:529-532, 1987), it was generally believed that plants in the family Gramineae could not be transformed with Agrobacterium (Potrykus I. Biotechnology 8:535-543, 1990).
Grimsley et al. (Nature 325:177-179, 1987) reported that cDNA from maize streak virus could be delivered to maize plants by Agrobacterium tumefaciens and that the plants became infected with the virus. The research did not demonstrate that the cDNA reached the maize genome nor did it demonstrate stable integration of streak virus nucleic acid. Later studies demonstrated that Agrobacterium could be used to deliver a kanamycin-resistance gene and a GUS (.beta.-glucuronidase) gene to shoot apices of maize after shoot apex injury (Gould J. et al. Plant Physiol. 95:426-434, 1991 and U.S. Pat. No. 5,177,010 to Goldman et al.). In these studies plants generated from the tissue exposed to Agrobacterium contained both transformed cells and non-transformed cells suggesting that the method did not uniformly deliver nucleic acid to the maize tissue.
European Patent Application Publication Number 604 662 A1 to Hiei et al. discloses a method for transforming monocotyledons using Agrobacterium. In this method, plant tissues were obtained from the monocotyledon maize and the tissues were exposed to Agrobacterium during the tissue dedifferentiation process. Hiei et al. disclose a maize transformation protocol using maize calli. Saito et al. disclose a method for transforming monocotyledons using the scutellum of immature embryos (European Application 672 752 A1). Ishida et al. also disclose a method specific for transforming maize by exposing immature embryos to A. tumefaciens (Nature Biotechnology, 1996, 14:745-750). The methods were optimized for inbred A188 maize lines. Transformation frequencies ranged from 12% to 30% at their highest for immature embryos from A188 lines that were 1.0-1.2 mm in length. Maize lines derived from crosses of A188 had significantly lower transformation frequencies ranging from 0.4% to about 5.3%. The transformation frequencies using A188 and A188 crosses are summarized in Table 1. A188 is not generally considered a commercially useful line and Ishida et al. failed to obtain recovery of stable transformants in lines other than those containing A188.
A need still exists for a method that will: (a) produce significantly higher transformation frequencies in lines other than those reported by Ishida et al. (supra); and, (b) produce transformed inbred lines other than line A188; including transformed inbreds representing a range of genetic diversities and having significant commercial utility.